الفهرس 
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Abstract
Hypericin has gained a great attention as a powerful photosensitizing and fluorescent agent for photodynamic therapy (PDT) and fluorescence diagnosis (FD) of cancer. However, the hydrophobic nature of hypericin and the tendency to form aggregates in aqueous environments constitute the most common obstruction restricting its biological applications. The reported studies about the photophysical properties of hypericin such as fluorescence, extinction coefficient, and quantum yield concluded that hypericin remains photoactive only while it is existing in the monomeric state. Hence, the aqueous formulae of hypericin are not biologically active unless they are chemically modified to remain in the monomeric state. In this thesis, different formulae of hypericin in aqueous media were prepared to improve the water solubility of hypericin for the different biological applications. Hypericin was first encapsulated with polyvinylpyrrolidone (hypericin@PVP) and the absorption and emission characteristics of hypericin@PVP in water were studied at room temperature. Also, the photostability of the prepared hypericin@PVP was studied under visible light irradiation. Upon encapsulation with PVP, hypericin possessed an improved solubility and stability in water as revealed by the relatively enhanced absorption and emission in water. The effect of the molecular weight of the PVP was assessed by adopting PVP with two different molecular weight, namely 10 and 360 KD. The absorbance and emission results confirm the efficient binding of the hypericin and the PVP with a binding constant (Kb) of 1.1x103and 1.2x105 M-1 for the two different PVP with molecular weights of 10 and 360 KD, respectively. Also, hypericin possessed improved fluorescence properties with a maximum emission enhancement of 212 and 200 fold relative to the hypericin in water in the case of 1x10-4 of 360 KD PVP and 1x10-2 M of 10 KD PVP, respectively. The results confirm that the association of hypericin and PVP in water led to the dissociation of aggregated hypericin into the monomeric state which is crucial for the effective photobiological implementation in PDT and FD. Moreover, the water-soluble hypericin@PVP demonstrated high photostability under visible light irradiation with an irradiance of 15 mW/cm2 and an exposure time up to 150 min. The improved absorption, emission, and photostability properties of hypericin in water is related to the promoting effect of the encapsulation of hypericin with PVP causing unique spectroscopic properties of the hypericin@PVP formula. Another formula to improve the emission properties of hypericin in aqueous solution using metallic nanoparticles was also studied. The formula constituted of c.a. 17 nm gold nanoparticles and hypericin that is encapsulated with PVP (hypericin@PVP-Au NPs). The hypericin@PVP-Au demonstrated effectively enhanced fluorescence properties with a maximum emission enhancement of 23 fold relative to that of hypericin@PVP formula. The enhanced emission arises from the metal-enhanced fluorescence (MEF) phenomenon observed when the organic fluorophores are present within the vicinity of metallic nanoparticles with an appropriate separation distance. The PVP acts to improve the solubility of hypericin in aqueous solution. It also acts as a spacer to separate the hypericin from gold nanoparticles with a suitable distance giving rise to enhanced emission of hypericin. In addition, the effect of the cellular environmental conditions on the aggregated hypericin in water (hypericin /H2O) was investigated. The results showed that the in vitro media mimicking the physiological conditions resulted in the effective dissociation of the hypericin aggregates into their monomeric state. This dissociation was associated with a significant increase of the emission intensity of hypericin. The photo-diagnostic and photodynamic properties of the different prepared formulae of hypericin, namely hypericin@PVP, hypericin@PVP-Au, hypericin/DMSO, and hypericin /H2O were evaluated in vitro, using confocal microscopy and MTT assay. The studies of the photo-diagnosis and the photodynamic action of the prepared aqueous hypericin formulae indicated that, under physiological conditions, hypericin undergoes spontaneous dissociation into the monomeric form which is necessary to exert its photoactivity. Therefore, the aggregation of hypericin in aqueous media can be overcome by using the biological fluids. Hypericin can be transformed into the monomeric form under the influence of the biological fluids such as protein binding. The MTT results of photocytotoxicity showed a larger mortality and a decreased the cell viability in the case of the cells treated with hypericin/DMSO, hypericin@PVP, and hypericin@PVP-Au compared to those treated with hypericin/H2O (p<0.01). The aqueous hypericin@PVP, and hypericin@PVP-Au formula possessed comparable PDT effects and they also demonstrated similar PDT efficacy to that of hypericin/DMSO using the same concentration of hypericin. Moreover, they showed enhanced efficacy for the FD, where brighter micrograph images could be obtained by using hypericin@PVP-Au even after a short incubation time of 1 h. The hypericin@PVP-Au with the underlying MEF holds a great promise for wide applications in cellular imaging and diagnosis.